Breast cancer is the second-leading cause of cancer death among American women (1). Approximately 25% of breast cancers overexpress the receptor tyrosine kinase (RTK) protein HER2, which induces cancer cell growth and survival (2). To combat HER2-positive breast cancer, researchers have developed therapies which inhibit HER2 functions, such as lapatinib. Response rates to lapatinib monotherapy are as high as 24% in previously untreated patients (3). Unfortunately, however, drug resistance develops in many patients who initially respond to lapatinib, and most patients do not show initial response. The mechanisms of this resistance are incompletely understood. Therefore, our objectives are to determine (a) the mechanism(s) of lapatinib resistance and (b) a therapeutic means to overcome it. By determining the mechanism of lapatinib resistance we will be empowered to provide additional treatment options for women with lapatinib-resistant breast cancer, for whom effective treatment options are lacking. To determine the mechanism of lapatinib resistance, we have developed a model of acquired lapatinib resistance by treating a HER2-positive breast cancer cell line with lapatinib for longer than 6 months (LapR cells), during which the cells became drug-resistant. This resistance is dependent on activation of PI3K through a novel mechanism. Our specific aims are to: (1) elucidate the mechanism of PI3K activation in these cells, (2) determine whether this mechanism is also involved in primary lapatinib resistance, in addition to acquired resistance, (3) devise a therapeutic strategy to overcome lapatinib resistance, and (4) determine whether the mechanism we have identified predicts patient outcomes. Aims 1, 2, and 3 will be achieved using biochemical, molecular, and cellular studies of in vitro cultures of cancer cell lines and mouse tumors. Aim 4 will be performed using patient data. These efforts will increase our knowledge about how some cancer cells escape lapatinib-induced death and how to eliminate such cells therapeutically, which may have significant impact on patients with HER2- positive breast cancer.